Modifications in the properties of nAChRs and excitatory synaptic transmission in neurons are thought to play an important role in the process of nicotine addiction in humans. However, the molecular mechanisms underlying behavioral responses to nicotine remain poorly understood. In this grant we propose to address these questions in a genetically accessible animal model. We have recently described the properties of nAChRs that mediate fast excitatory transmission in cultured Drosophila neurons. These cultures provide a unique opportunity to explore the cellular and molecular mechanisms important in regulation of nAChRs and cholinergic transmission at central synapses. Our first aim focuses on characterizing the biophysical, pharmacological, and molecular properties of nAChRs mediating nicotine-evoked currents/calcium transients in Drosophila neurons. In addition we will determine the function of different nAChR genes by examining the consequences of knocking out or reducing the expression of specific subunit/s in the cultured neurons. This will be accomplished by examining neurona! responses to nicotine in cultures prepared from deficiencies uncovering nAChR genes or in cultures exposed to dsRNA targeted to particular nAChR subunit/s. The second aim examines the role of nicotine in regulation of nAChRs and cholinergic transmission. Neuronal responses to nicotinefACh, 1125-aBTX binding, and the properties of cholinergic transmission will be monitored in neurons chronically exposed to nicotine. We will also examine the effects of short duration, repetitive treatments with nicotine, mimicking the exposure that occurs during smoking. The cAMP signaling cascade plays a role in regulating responses to ethanol and cocaine in both flies and mammals. In our third aim we will examine the properties of nAChRs and cholinergic synaptic currents in cAMP signaling mutants to determine if the effects of nicotine are also modulated by CAMP. Finally, we will use differential display and cDNA microarrays to identify genes that are differentially regulated by nicotine. The results of our studies will provide important insights into the mo!ecular mechanisms underlying nicotine-induced regulation of nAChRs. These data will also contribute to the general understanding of the molecular mechanisms important in regulating transmission at excitatory synapses, likely to be highly conserved between vertebrates and invertebrates. Finally, this knowledge may be useful in the design of drugs ancVor therapies targeted at ameliorating or preventing nicotine addiction in humans by regulating nAChRs and synaptic function.